EP0735143B1

EP0735143B1 - Process for preparing D-amino acids

Info

Publication number: EP0735143B1
Application number: EP96301872A
Authority: EP
Inventors: Masakatsu Furui; Eiji Takahashi; Hiroyasu Seko; Takeji Shibatani
Original assignee: Tanabe Seiyaku Co Ltd
Current assignee: Tanabe Seiyaku Co Ltd
Priority date: 1995-03-28
Filing date: 1996-03-19
Publication date: 1998-11-18
Anticipated expiration: 2016-03-19
Also published as: DE69600978D1; EP0735143A1; DE69600978T2; US5783427A

Description

BACKGROUND OF THE INVENTION

This invention relates to a novel process for preparing the D-amino acids D-valine, D-leucine and D-isoleucine utilizing microorganisms.

The D-Amino acids D-valine, D-leucine and D-isoleucine are useful compounds as starting materials or synthetic intermediates for preparation of various medicines such as antibiotics, or optically resolving agents. In the prior art, as a process for preparing these amino acids, there have been known a fractional crystallization method of a racemic material, an optical resolution method by chromatography and a physicochemical method such as an organochemical asymmetric synthesis and the like. As a biochemical method, there have been known a method of asymmetrically hydrolyzing N-acetyl-DL-amino acids by using a microorganism enzyme (Applied and Environmental Microbiology, vol. 54, pp. 984-989 (1988)), a method of asymmetrically hydrolyzing 5-methylthioethylhydantoin, 5-isopropylhydantoin, 5-isopentanoylhydantoin or 5-sec-butylhydantoin by using a microorganism enzyme (Journal of Fermentation Technology, vol. 56, pp. 492-498 (1978)), a method of asymmetrically hydrolyzing 5-(4-imidazolemethyl)-hydrantoin by using a microorganism enzyme (Agric. Biol. Chem., vol. 51, pp. 715-719 (1987)), a method of hydrolyzing D-N-carbamoyl-α-amino acids (PCT Patent Publication No. WO 92/10579), a method of asymmetrically hydrolyzing DL-aminonitrile by using a microorganism enzyme (Bull. Inst. Chem. Res., Kyoto Univ., vol. 65, pp. 141-143 (1987)) or a method of transferring an amino group of an α-keto acid by using a microorganism enzyme (J. Biotechnol., vol. 8, pp. 243-248 (1988)).

In the above-mentioned physicochemical method, there are disadvantages that operation is complicated or troublesome, and yield and optical purity of the product are low. In the biochemical method, there are disadvantages that 5-methylthioethylhydantoin, 5-isopropylhydantoin, 5-isopentanoylhydantoin, 5-sec-butylhydantoin, 5-(4-imidazole-methyl)-hydrantoin and D-N-carbamoyl-α-amino acids which are used as a substrate are expensive, separation of the product is difficult and regeneration of coenzyme is required. Thus, it has been desired to develop a process for preparing a D-amino acid which solves at least one problem as mentioned above.

In JP-7015433 there is described a fermentation process in which DL-isomers of α-phenylglycine, methionine and threonine are used in order to obtain their D-isomers.

SUMMARY OF THE INVENTION

The present inventors have studied intensively and consequently found microorganisms having ability to selectively degrade only a L-isomer in a racemic amino acid, to accomplish the present invention.

That is, the present invention is a process for preparing a D-amino acid selected from the group consisting of D-valine, D-leucine and D-isoleucine which comprises the steps of:

making a culture or treated culture of a microorganism having ability to asymmetrically degrade an L-amino acid selected from the group consisting of L-valine, L-leucine and L-isoleucine act on a racemic amino acid corresponding to said L-amino acid; and

separating and collecting the remaining D-amino acid;

wherein the micro-organism belongs to Proteus, Providencia or Yarrowia, said culture of the microorganism is a culture broth or a living cell, and said treated culture of the microorganism is a culture supernatant, a washed cell, a dried cell, a ground cell, an autolysate of a cell, an extract of a cell, a partially purified enzyme or a purified enzyme.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following, the present invention is explained in detail.

In the present specification, the term "amino acid" means a specific amino acid selected from the group consisting of valine, leucine and isoleucine.

The racemic amino acid to be used as a starting material in the present invention may be not only one containing equal amounts of D-isomer and L-isomer, but one containing both of these optical isomers in any ratio of mixture. As the racemic amino acid, racemic modifications of valine, leucine and isoleucine are used.

As a microorganism degrading L-valine, L-leucine or L-isoleucine, there may be mentioned, for example, microorganisms belonging to Achromobacter, Proteus, Providencia or Yarrowia. Among them, preferred are microorganisms belonging to Proteus or Providencia, and particularly preferred are microorganisms belonging to Proteus (e.g., Proteus vulgaris).

Specific examples of these microorganisms include Achromobacter liquidum OUT 8012 (FERM P-12684), Proteus vulgaris RIMD KS (IAM 12003), Proteus vulgaris AHU 1469, Proteus vulgaris AHU 1472, Proteus vulgaris AHU 1474, Providencia alcalifaciens JCM 1673, Providencia rettgeri ATCC 25932, Yarrowia lipolytica IFO 0717, Yarrowia lipolytica IFO 0746, Yarrowia lipolytica IFO 1195, Yarrowia lipolytica IFO 1209, Yarrowia lipolytica IFO 1548 or the like.

The microorganism to be used in the present invention may be a strain which is newly separated from soil, food, an animal or the like so long as it has an ability necessary for the present invention. Further, there may be used a mutant obtained by artificial treatment such as irradiation of UV ray or treatment using a mutating agent or a strain derived from the above microorganism by a genetic engineering means such as recombination of DNA or cell fusion, or bioengineering means. For example, according to the genetic engineering means, a gene of an objective enzyme is isolated from a chromosome fragment of a microorganism producing an enzyme which has ability to asymmetrically degrade a L-amino acid, then a recombinant plasmid obtained by introducing the gene into an appropriate plasmid vector is formed, and an appropriate host microorganism is transformed by the recombinant plasmid to obtain a microorganism having ability to produce an enzyme or having an improved productivity of said enzyme. Further, a host microorganism having other excellent characteristics (e.g., easy in culture or the like) may be transformed by the recombinant plasmid, if necessary.

The culture or treated culture of the microorganism to be used in the present invention is any culture or treated culture so long as it has ability to asymmetrically degrade at least one of the above specific L-amino acids. As the culture, there may be mentioned, for example, a culture broth or a living cell, and as the treated culture, there may be mentioned, for example, a treated culture broth such as a culture supernatant, a treated cell such as a washed cell, a dried cell, a ground cell, an autolysate of a cell, an extract of a cell, or a partially purified enzyme or purified enzyme obtained therefrom according to the conventional manner.

The above culture (e.g., a culture broth, living cell or the like) can be obtained by, for example, culturing the microorganism in a medium (e.g., a conventional medium containing carbon source, nitrogen source and an inorganic salt), at pH about 5 to pH about 8 at ordinary temperature to under heating (preferably about 20 °C to 40 °C) and under aerobic conditions. Further, during culture, by adding about 0.001 % to about 10 %, preferably about 0.1 % to about 2 %, particularly about 0.1 % to about 1 % of an appropriate amino acid to the medium, the desired enzyme activity can be enhanced.

The living cell and the culture supernatant can be prepared from the thus-obtained culture broth as described above, by means of such as centrifugation or filtration. The washed cell can be obtained by washing a living cell with a physiological saline, and the dried cell can be obtained by subjecting a living cell or a washed cell to lyophilization or acetone drying. The ground cell can be obtained by treating a living cell or a washed cell by various known physicochemical methods, for example, ultrasonication, French press, osmotic pressure, freezing and thawing, alumina grinding or lysokinase, a surfactant or an organic solvent. The extract of a cell can be obtained by, for example, removing insoluble matters from a ground cell by filtration or centrifugation. The partially purified enzyme or purified enzyme can be obtained by, for example, fractionating an enzyme from a fraction of a pulverized cell or a culture supernatant according to a conventional manner such as fractionation using ammonium sulfate, ion exchange chromatography or gel filtration chromatography and purifying the enzyme by using ability to selectively degrade the above specific L-amino acid as an index.

The above microorganism cells, treated cells or enzymes may be used as such and may be used after immobilizing it by a polyacrylamide method, a sulfur-containing polysaccharide gel method (e.g., a carrageenan gel method), an alginic acid gel method, an agar gel method or the like.

Also, the microorganism which can asymmetrically degrade the L-amino acid is cultured in a medium containing the racemic amino acid and the D-amino acid remaining in the medium may be separated and collected.

The asymmetric degradation according to the present invention can be carried out by bringing the racemic amino acid which is a starting compound into contact with the culture or treated culture of the microorganism having ability to asymmetrically degrade the L-amino acid in a solution, followed by incubation. Further, if desired, the reaction may be carried out concurrently with culturing the micro-organism. In such a case, the reaction can be carried out by using a medium to which the racemic amino acid is previously added under the same conditions as those of culture.

The reaction can be carried out suitably in an aqueous solution. Further, the reaction proceeds suitably at ordinary temperature to under heating, preferably about 10 °C to about 50 °C, particularly preferably about 25 °C to about 40 °C. It is preferred to adjust the pH of the reaction mixture to pH about 5 to pH about 11, particularly pH about 6 to pH about 9.

The charged concentration (w/v) of the racemic amino acid which is a starting compound to be used as a reaction substrate is generally preferably about 0.05 % to about 30 %, particularly about 1 % to about 20 %. The starting compound may be added at one time in the beginning or may be added several times with divided amounts during the reaction.

When the living cell is used in the present invention, it is preferred to add a surfactant to the reaction mixture since the reaction time can be shortened. As an example of the surfactant to be used for the above purpose, there may be mentioned cetyl pyridinium bromide, cetyl trimethylammonium bromide or p-isooctylphenyl ether (Triton X-100, trade name, produced by Rohm & Haas Co., U.S.A.), and it is preferred to use the surfactant in an amount of about 0.0001 % to about 0.1 % based on the amount of the reaction mixture.

After completion of the reaction, collection and isolation of the D-amino acid from the reaction mixture can be carried out easily according to the conventional manner. For example, after insoluble materials such as a cell are removed from the reaction mixture by centrifugation, the reaction mixture is treated with activated carbon to adsorb and remove a dye or the like and the mixture is concentrated under reduced pressure. Thereafter, the reaction mixture is subjected to crystallization under cooling to obtain crystals of the D-amino acid.

Detection whether or not the culture or the treated culture of the microorganism has ability to asymmetrically degrade the L-amino acid can be carried out easily according to the above reaction method, for example, as described below. That is, the culture or the treated culture of the microorganism to be detected is added to a medium or aqueous solution containing the racemic amino acid, and the mixture is shaken at 30 °C for 120 hours. The solution after completion of the reaction is analyzed and quantitated by high performance liquid chromatography using an optically active column (e.g., CROWNPAK CR(+), trade name, manufactured by Daicel Kagaku Kogyo Co. or SUMICHIRAL OA-5000, trade name, manufactured by Sumika Analysis Center) to measure the respective contents of the D-amino acid and the L-amino acid. By the measurement, for example, when it is found that a L-isomer is reduced and a D-isomer remains in the reaction mixture, it is judged that the culture or the treated culture of the microorganism has ability to asymmetrically degrade the L-amino acid.

EXAMPLES

The present invention is described in detail by referring to Examples, but should not be construed to be limited thereto.

In the present specification, "%" always means "weight/ volume (g/dl)". Further, in Examples, quantitation of an optical isomer of methionine was carried out by high performance liquid chromatography using CROWNPAK CR(+) (trade name, manufactured by Daicel Kagaku Kogyo Co.) and other amino acids by high performance liquid chromatography using SUMICHIRAL OA-5000 (trade name, manufactured by Sumika Analysis Center).

Example 1

Into a test tube was charged 3 ml of a medium (pH 7.0) comprising 2 % of DL-valine, 0.5 % of ammonium sulfate, 0.1 % of potassium dihydrogen phosphate, 0.05 % of magnesium sulfate and 0.02 % of a yeast extract, and the medium was sterilized at 120 °C for 10 minutes. Into the medium were inoculated microorganisms in Table 1 shown below, respectively. After the microorganisms were cultured while shaking at 30 °C for 144 hours, D-valine remaining in the culture broth was quantitated. The contents of D-valine were as shown in Table 1. Further, almost no L-valine which was an antipode was detected from the culture broth.

Name of strain	Remaining D-valine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	3.8
Proteus vulgaris RIMD KS (IAM 12003)	7.3
Proteus vulgaris AHU 1469	8.3
Proteus vulgaris AHU 1472	8.0
Proteus vulgaris AHU 1474	7.1

Example 2

Into a shaking flask having a volume of 500 ml was charged 100 ml of a medium (pH 7.0) comprising 0.5 % of DL-methionine, 1.0 % of polypeptone, 1.0 % of a yeast extract and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. A loopful of Proteus vulgaris RIMD KS (IAM 12003) was inoculated into the medium, and cultured at 30 °C for 20 hours with shaking. The cells collected by centrifuging 1600 ml of the above culture broth were suspended in a physiological saline and the suspension was further centrifuged to collect cells. To the cells was added 800 ml of a 50 mM phosphate buffer (pH 7.0) containing 5 % of DL-valine, and the mixture was reacted at 30 °C for 72 hours to effect asymmetric degradation to completely degrade L-valine. After the reaction, the mixture was sterilized by centrifugation to obtain a supernatant. Ultrafiltration was carried out in order to remove protein and others in the above supernatant, whereby a filtrate was obtained. The filtrate was concentrated under reduced pressure, and the concentrate was crystallized by cooling to obtain 4.C g of crystals of D-valine.

Optical rotation: [α] 20 / D : -27.5° (C = 8, 6N HCl)

Optical purity: 100 %

Example 3

Into 3 ml of the sterilized medium (pH 7.0) comprising 0.5 % of DL-valine, 1.0 % of polypeptone, 1.0 % of a yeast extract and 0.5 % of sodium chloride were inoculated microorganisms in Table 2 shown below, respectively, and cultured at 30 °C for 20 hours with shaking. Cells collected by centrifuging 3 ml of the above culture were suspended in a physiological saline and the suspension was further centrifuged to collect cells. To the cells was added 2 ml of a 50 mM phosphate buffer (pH 7.0) containing 5 % of DL-valine, and the mixture was reacted at 30 °C for 144 hours to effect asymmetric degradation. The contents of D-valine were as shown in Table 2. Further, almost no L-valine which was an antipode was detected from the reaction mixture.

Name of strain	Remaining D-valine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	19.5
Proteus vulgaris RIMD KS (IAM 12003)	20.8
Proteus vulgaris AHU 1469	20.6
Proteus vulgaris AHU 1472	15.2
Proteus vulgaris AHU 1474	22.5
Providencia alcalifaciens JCM 1673	22.3
Providencia rettqeri ATCC 25932	19.2
Yarrowia lipolytica IFO 0717	23.7
Yarrowia lipolytica IFO 1195	22.2
Yarrowia lipolytica IFO 1209	19.4

Example 4

Into a test tube was charged 3 ml of a medium (pH 7.0) comprising 1 % of DL-leucine, 1 % of a yeast extract, 1 % of polypeptone and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. Into the medium were inoculated microorganisms in Table 3 shown below, respectively. After the microorganisms were cultured while shaking at 30 °C for 24 hours, D-leucine remaining in the culture broth was quantitated. The contents of D-leucine were as shown in Table 3. Further, almost no L-leucine which was an antipode was detected from the culture broth.

Name of strain	Remaining D-leucine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	4.5
Proteus vulgaris RIMD KS (IAM 12003)	4.2
Proteus vulgaris AHU 1469	5.0
Proteus vulgaris AHU 1472	5.0
Proteus vulgaris AHU 1474	3.2
Yarrowia lipolytica IFO 1548	5.0
Yarrowia lipolytica IFO 1209	5.0

Example 5

Into a shaking flask having a volume of 500 ml was charged 100 ml of a medium (pH 7.0) comprising 0.5 % of DL-methionine, 1.0 % of polypeptone, 1.0 % of a yeast extract and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. A loopful of Proteus vulgaris RIMD KS (IAM 12003) was inoculated into the medium and cultured at 30 °C for 20 hours with shaking. The cells collected from 1600 ml of the above culture broth by centrifugation were suspended in a physiological saline and then the suspension was further centrifuged to collect cells. To the cells was added 800 ml of a 50 mM phosphate buffer (pH 7.0) containing 5 % of DL-leucine, and the mixture subjected to asymmetric degradation at 30 °C for 72 hours to degrade L-leucine completely. After the reaction, the cells were removed by centrifugation, and subsequent procedures were carried out in the same manner as in Example 2 to obtain 5.3 g of D-leucine.

Optical rotation: [α] 20 / D : -15.3° (C = 4, 6N HCl)

Optical purity: 100 %

Example 6

Into a test tube was charged 3 ml of a medium (pH 7.0) comprising 1 % of DL-leucine, 1 % of a yeast extract, 1 % of polypeptone and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. Into the medium were inoculated microorganisms in Table 4 shown below, respectively. After the microorganisms were cultured while shaking at 30 °C for 24 hours, cells collected by centrifugation were suspended in a physiological saline and the suspension was further centrifuged to collect cells. To the cells was added 2 ml of a 50 mM phosphate buffer (pH 7.0) containing 1 % of DL-leucine, and the mixture was reacted at 30 °C for 24 hours to effect asymmetric degradation. The contents of D-leucine were as shown in Table 4. Further, almost no L-leucine which was an antipode was detected from the reaction mixture.

Name of strain	Remaining D-leucine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	4.1
Proteus vulgaris RIMD KS (IAM 12003)	4.5
Proteus vulgaris AHU 1469	4.5
Proteus vulgaris AHU 1472	4.0
Proteus vulgaris AHU 1474	4.7
Providencia alcalifaciens JCM 1673	4.4
Yarrowia lipolytica IFO 0717	1.8

Example 7

Into a test tube was charged 3 ml of a medium (pH 7.0) comprising 1 % of DL-isoleucine, 1 % of a yeast extract, 1 % of polypeptone and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. Into the medium were inoculated microorganisms in Table 5 shown below, respectively. After the microorganisms were cultured while shaking at 30 °C for 24 hours, D-isoleucine remaining in the culture broth was quantitated. The contents of D-isoleucine were as shown in Table 5. Further, almost no L-isoleucine which was an antipode was detected from the culture broth.

Name of strain	Remaining D-iso-leucine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	3.9
Proteus vulqaris RIMD KS (IAM 12003)	3.4
Proteus vulgaris AHU 1469	3.6
Proteus vulgaris AHU 1472	3.5
Proteus vulgaris AHU 1474	3.9
Providencia alcalifaciens JCM 1673	3.5
Yarrowia lipolytica IFO 1209	3.4

Example 8

Into a shaking flask having a volume of 500 ml was charged 100 ml of a medium (pH 7.0) comprising 0.5 % of DL-methionine, 1,0 % of polypeptone, 1.0 % of a yeast extract and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. A loopful of Proteus vulgaris RIMD KS (IAM 12003) was inoculated into the medium, and cultured at 30 °C for 20 hours with shaking. The cells collected from 1600 ml of the above culture broth by centrifugation were suspended in a physiological saline and then the suspension was further centrifuged to collect cells. To the cells was added 800 ml of a 50 mM phosphate buffer (pH 7.0) containing 5 % of DL-isoleucine, and the mixture subjected to asymmetric degradation at 30 °C for 72 hours to degrade L-isoleucine completely. After the reaction, the cells were removed by centrifugation, and subsequent procedures were carried out in the same manner as in Example 2 to obtain 8.5 g of D-isoleucine.

Optical rotation: [α] 20 / D : -38.0° (C = 4, 6N HCl)

Optical purity: 100 %

Example 9

Into a test tube was charged 3 ml of a medium (pH 7.0) comprising 1 % of DL-isoleucine, 1 % of a yeast extract, 1 % of polypeptone and 0.5 % of sodium chloride, and the medium was sterilized at 120 °C for 10 minutes. Into the medium were inoculated microorganisms in Table 6 shown below, respectively. After the microorganisms were cultured while shaking at 30 °C for 24 hours, cells collected by centrifugation were suspended in a physiological saline and the suspension was further centrifuged to collect cells. To the cells was added 2 ml of a 50 mM phosphate buffer (pH 7.0) containing 2 % of DL-isoleucine, and the mixture was reacted at 30 °C for 24 hours to effect asymmetric degradation. The contents of D-isoleucine were as shown in Table 6. Further, almost no L-isoleucine which was an antipode was detected from the reaction mixture.

Name of strain	Remaining D-isoleucine (mg/ml)
Achromobacter liquidum OUT 8012 (FERM P-12684)	8.1
Proteus vulgaris AHU 1469	9.0
Proteus vulgaris AHU 1472	8.9
Proteus vulgaris AHU 1474	9.2
Providencia alcalifaciens JCM 1673	9.0
Yarrowia lipolytica IFO 0717	9.6
Yarrowia lipolytica IFO 1548	8.6
Yarrowia lipolytica IFO 0746	7.9
Yarrowia lipolytica IFO 1195	8.6
Yarrowia lipolytica IFO 1209	4.8

As described above, according to the process of the present invention, the D-amino acids can be prepared industrially from an inexpensive racemic amino acids with extremely good efficiency and high optical purity. Thus, the process of the present invention is an industrially advantageous preparation process.

Claims

A process for preparing a D-amino acid selected from the group consisting D-valine, D-leucine and D-isoleucine which comprises the steps of:

making a culture or treated culture of a microorganism having ability to asymmetrically degrade an L-amino acid selected from the group consisting of L-valine, L-leucine and L-isoleucine act on a racemic amino acid corresponding to said L-amino acid; and

separating and collecting the remaining D-amino acid;

wherein the micro-organism belongs to Proteus, Providencia or Yarrowia, said culture of the microorganism is a culture broth or a living cell, and said treated culture of the microorganism is a culture supernatant, a washed cell, a dried cell, a ground cell, an autolysate of a cell, an extract of a cell, a partially purified enzyme or a purified enzyme.
The process according to claim 1, wherein the micro-organism having ability to asymmetrically degrade an L-aminoacid selected from the group consisting of L-valine, L-leucine and L-isoleucine is a micro-organism belonging to Proteus vulgaris.
The process according to claim 1, wherein the micro-organism having ability to asymmetrically degrade an L-amino acid selected from the group consisting of L-valine, L-leucine and L-iscleucine is at least one microorganism selected from the group consisting of Proteus vulgaris RIMO KS (IAM 12003). Providencia alcalifaciens JCM 1673, Providencia rettgeri ATCC 25932, Yarrowia lipolytica IFO 0717, Yarrowia lipolytica IFO 0746, Yarrowia lipolytica IFO 1195, Yarrowia lipolytica IFO 1209 and Yarrowia lipolytica IFO 1548.
The process according to claim 1, wherein the step of making a culture or treated culture of a microorganism having ability to asymmetrically degrade the L-amino acid act on a racemic amino acid is carried out in a medium concurrently with culturing the microorganism having ability to asymmetrically degrade the L-amino acid.
The process according to claim 1, wherein making a culture or treated culture of a microorganism having ability to asymmetrically degrade the L-amino acid act on a racemic amino acid is carried out in an aqueous solution at about 10°C to about 50°C at pH about 5 to pH about 11.
The process according to claim 1, wherein making a culture or treated culture of a microorganism having ability to asymmetrically degrade the L-amino acid act on a racemic amino acid is carried out in an aqueous solution at about 25°C to about 40°C at pH about 6 to pH about 9.
The process according to claim 1, wherein the racemic amino acid is added to the culture medium in an amount of about 0.05% to about 30% in terms of w/v.
The process according to claim 1, wherein the racemic amino acid is added to the culture medium in an amount of about 1% to about 20% in terms of w/v.